Potency Evaluations of Recombinant Botulinum Neurotoxin A1 Mutants Designed to Reduce Toxicity
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Published:2024-08-17
Issue:16
Volume:25
Page:8955
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ISSN:1422-0067
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Container-title:International Journal of Molecular Sciences
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language:en
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Short-container-title:IJMS
Author:
Viravathana Polrit1, Tepp William H.1, Bradshaw Marite1, Przedpelski Amanda2, Barbieri Joseph T.2, Pellett Sabine1ORCID
Affiliation:
1. Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA 2. Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
Abstract
Recombinant mutant holotoxin BoNTs (rBoNTs) are being evaluated as possible vaccines against botulism. Previously, several rBoNTs containing 2–3 amino acid mutations in the light chain (LC) showed significant decreases in toxicity (2.5-million-fold–12.5-million-fold) versus wild-type BoNT/A1, leading to their current exclusion from the Federal Select Agent list. In this study, we added four additional mutations in the receptor-binding domain, translocation domain, and enzymatic cleft to further decrease toxicity, creating 7M rBoNT/A1. Due to poor expression in E. coli, 7M rBoNT/A1 was produced in an endogenous C. botulinum expression system. This protein had higher residual toxicity (LD50: 280 ng/mouse) than previously reported for the catalytically inactive rBoNT/A1 containing only three of the mutations (>10 µg/mouse). To investigate this discrepancy, several additional rBoNT/A1 constructs containing individual sets of amino acid substitutions from 7M rBoNT/A1 and related mutations were also endogenously produced. Similarly to endogenously produced 7M rBoNT/A1, all of the endogenously produced mutants had ~100–1000-fold greater toxicity than what was reported for their original heterologous host counterparts. A combination of mutations in multiple functional domains resulted in a greater but not multiplicative reduction in toxicity. This report demonstrates the impact of production systems on residual toxicity of genetically inactivated rBoNTs.
Funder
Food and Research Institute of the University of Wisconsin–Madison, the National Institute of Health, and the National Institute of Allergy and Infectious Disease
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